Fluorescent detection of protein kinase based on zirconium ions-immobilized magnetic nanoparticles

• Present a novel method for PKA activity analysis based on magnetic separation.
• Zr-NTA MNPs are implemented in detection of PKA activation in cell lysates.
• Specific adsorption between zirconium ions and phosphate group.
• Highly selective, sensitive and fast recognition of phosphorylated peptides.
• The MNPs are recyclable.

We report here an affinity separation-based fluorometric method for monitoring the activity and inhibition of protein kinase. In this assay, when the fluorescein isothiocyanate (FITC) labeled substrate peptides (S-peptide) are phosphorylated by kinase, the product peptides (P-peptide) will be adsorbed and concentrated onto the surface of Zr4+-immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Zr-NTA MNPs) through the chelation of Zr4+ and phosphate groups. After magnetic separation, the fluorescence intensity of the homogeneous solution changes dramatically. Hence the fluorescence response allows this MNPs-based method to easily probe kinase activity by a spectrometer. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.5 mU μL−1). Moreover, the system is successfully applied to estimate the IC50 value of PKA inhibitor H-89 and detect the Forskolin/3-isobutyl-1-methylxanthine (IBMX) stimulated activation of PKA in cell lysate. Additionally, Zr-NTA MNPs are reusable by stripping Zr4+ ions from NTA-coated MNPs and rechelating again. This method, which relies on the surface-functionalized MNPs, presents a promising candidate for simple and cost-effective assay of kinase activity and inhibitor screening.

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